Chitinase genes responsive to cold encode antifreeze proteins in winter cereals

Abstract

Antifreeze proteins similar to two different chitinases accumulate during cold acclimation in winter rye (Secale cereale). To determine whether these cold-responsive chitinases require post-translational modification to bind to ice, cDNAs coding for two different full-length chitinases were isolated from a cDNA library produced from cold-acclimated winter rye leaves. CHT9 is a 1,193-bp clone that encodes a 31.7-kD class I chitinase and CHT46 is a 998-bp clone that codes for a 24.8-kD class II chitinase. Chitinase-antifreeze proteins purified from the plant were similar in mass to the predicted mature products of CHT9 and CHT46, thus indicating that there was little chemical modification of the amino acid sequences in planta. To confirm these results, the mature sequences of CHT9 and CHT46 were expressed in Escherichia coli and the products of both cDNAs modified the growth of ice. Transcripts of both genes accumulated late in cold acclimation in winter rye. Southern analysis of winter rye genomic DNA indicated the presence of a small gene family homologous to CHT46. In hexaploid wheat, CHT46 homologs mapped to the homeologous group 1 chromosomes and were expressed in response to cold and drought. We conclude that two novel cold-responsive genes encoding chitinases with ice-binding activity may have arisen in winter rye and other cereals through gene duplication.

Figure 1

The nucleotide and deduced amino acid sequences of CHT9. The nucleotide sequence (1,193 bp) is numbered relative to the first nucleotide in the translational start codon ATG and the stop codon is indicated by an asterisk. The predicted amino acid sequence is shown directly below the nucleotide sequence with the predicted first amino acid of the mature protein shown in bold and the signal sequence shown in italics. The region used for the gene-specific probe (30-mer oligonucleotide) is underlined.

Figure 2

The nucleotide and deduced amino acid sequences of CHT46. The nucleotide sequence (998 bp) is numbered relative to the first nucleotide in the translational start codon ATG and the stop codon is indicated by an asterisk. The predicted amino acid sequence is shown directly below the nucleotide sequence with the predicted first amino acid of the mature protein shown in bold and the signal sequence shown in italics. The region used for the gene-specific probe (a fragment generated by XhoI digestion) is underlined.

Figure 3

Multiple sequence alignment of amino acid sequences for chitinases from rye and other species. The predicted amino acid sequences for CHT9 and CHT46 were aligned with the barley chitinase HvCHT2a (T. Bryngelsson, personal communication, GenBank accession no. X78671), a tobacco chitinase (Shinshi et al., 1990; GenBank accession no. X16939), and a Chinese spring wheat chitinase (Liao et al., 1994, GenBank accession no. X76041). Identical amino acids are highlighted on a black background, similar amino acids are shown on a gray background, and dashes indicate gaps. Within the mature proteins, amino acids that differ between tobacco, which lacks antifreeze activity, and both CHT9 and CHT46, which exhibit antifreeze activity, are underlined.

Figure 4

Molecular masses of apoplastic CHT-AFPs purified from CA winter rye leaves determined by mass spectrometry. The two rye CHT-AFPs were purified by column chromatography and their identities were confirmed by SDS-PAGE, immunoblotting, and the presence of antifreeze activity. Samples containing 0.8 μg of protein were examined by mass spectrometry. Raw data (m/z) were processed with the MaxEnt algorithm of MassLynx 2.0 to yield spectra on a true molecular mass scale. A, The molecular mass of the 35-kD CHT-AFP was 31,693 ± 7 D. B, The molecular mass of the 28-kD CHT-AFP was 24,919 ± 3 D.

Figure 5

Expression of CHT9 and CHT46 in winter rye during cold acclimation and deacclimation. Total RNA was isolated from the youngest leaf tissue of plants at each time point. The RNA (10 μg) was denatured at 65°C for 15 min, separated in a formaldehyde 1.4% (w/v) agarose gel, and stained with ethidium bromide. The 25S rRNA of the ethidium-stained gel is shown above. The RNA was transferred to a nylon membrane and hybridized with radiolabeled gene-specific probes for CHT9 and CHT46. The sizes of the transcripts were estimated using the RNA standard markers as indicated on the left. A, Winter rye plants were grown under cold-acclimating conditions for 1, 3, 5, and 7 weeks. Neither chitinase gene was expressed immediately in plants transferred to cold temperature. Instead, CHT9 was detected as a 1.25-kb transcript and CHT46 was detected as a 1.00-kb transcript in leaves only after the plants had been cold acclimated for at least 5 weeks. B, Plants that had been cold acclimated for 7 weeks were transferred back to 20°C for 6, 12, 30, and 48 h to deacclimate. The transcripts for CHT9 and CHT46 were not detectable within 30 h of transferring the plants to 20°C, thus indicating that expression of these genes is responsive to cold temperature.

Figure 6

Expression of pET12α/CHT9-12 in E. coli BL21 cells and antifreeze activity of CHT9. A, Lanes 1 through 4 show the accumulation of the 35-kD CHT9 (arrowhead) in the periplasmic fraction of cells where expression of pET12α/CHT9-12 was induced by IPTG and the culture was incubated at 25°C for 0, 3, 24, and 48 h. Periplasmic proteins (20 μg lane⁻¹) isolated from the cells at each time point were solubilized and separated by SDS-PAGE on a 12% (w/v) polyacrylamide gel stained with Coomassie Blue. The molecular masses of Bio-Rad protein standards are shown on the left. B, Antifreeze activity was assayed by observing the growth of ice crystals in solutions of the periplasmic fraction isolated at each time point. The crystals are shown with the basal plane (a-axes) parallel to the plane of the page and the c-axis perpendicular to the page. The total protein concentration in each solution was adjusted to 1.2 mg mL⁻¹. The formation of hexagonally shaped ice crystals was observed 24 h after induction of pET12α/CHT9-12 expression. This increase in antifreeze activity was correlated with the accumulation of a 35-kD protein in the periplasm.

Figure 7

Purification and antifreeze activity of CHT46 produced by expressing OmpA/His6/CHT46 in E. coli JM105 cells. A, As shown in the left lane, OmpA-His6-CHT46 (1.2 μg) was purified from cell lysates, solubilized, and separated on a SDS-polyacrylamide gel (12%, w/v) stained with Coomassie Blue. The right-hand lane shows the presence of CHT46 after thrombin cleavage of OmpA-His6-CHT46. The positions of Bio-Rad broad range prestained molecular mass protein standards (center lane) are shown on the left. B, After cleavage of the OmpA leader and His-tag, hexagonally shaped ice crystals grew during freezing of CHT46 solutions containing 0.06 mg of total soluble protein mL⁻¹ (ice crystal shown with c-axis perpendicular to the plane of the page). C, After concentrating the solution containing CHT46 to 1.35 mg of total soluble protein mL⁻¹, the ice crystals grew to form hexagonally shaped columns (in the crystal shown, the c-axis is parallel to the plane of the page). The increased c-axis growth of the ice crystal indicates a greater amount of antifreeze activity.

Figure 8

Estimation of gene copy number and mapping of CHT46 homologs in winter cereals. DNA (1.8 μg) from winter rye cultivars Musketeer (MUSK) and Puma (PUMA), from the wheat cultivar Chinese Spring (CS), and from the group 1 chromosome ditelocentric series of Chinese Spring (1AS, 1AL, 1BS, 1BL, 1DS, and 1DL) were digested with XbaI. The last wash after hybridization of the membranes was in 0.2× SSC containing 0.1% (w/v) SDS at 68°C for 1 h. Numbers at right indicate estimated lengths of the fragments in kilobases.

Figure 9

Developmental, environmental, and hormonal regulation of expression of CHT46 homologs in winter wheat. Total RNA was isolated from the winter wheat cv Frederick grown at 20°C for 7 d (NA) at 5°C for 1, 6 and 36 d (CA1, CA6, and CA36, respectively), and deacclimated at 20°C for 1 d (DA). To examine tissue specificity, total RNA was isolated from CA36 plants divided into leaves, crowns, and roots. In addition, RNA was isolated from plants subjected to heat shock, salt stress, drought stress, low light (250 μmol photons m⁻² s⁻¹), and high light (800 μmol photons m⁻² s⁻¹), as well as plants treated with ABA. Northern blots were probed with pCHT46 at high stringency. There was a low level of chitinase gene expression in NA, CA1, root, heat shock, salt, ABA, low light, and high light plants. Genes that hybridized to CHT46 were up-regulated by cold (CA) and drought in leaves and crowns. The hybridization signal produced by CA plants was significantly reduced after transferring the plants back to 20°C to deacclimate (DA).